Background. Glufosfamide (GLU) is a glucose conjugate of ifosfamide in which isophosphoramide mustard is glycosidically linked to the β-D-glucose molecule. Based on GLU structure, it is considered a targeted chemotherapy with fewer side effects. The main objective of the current study is to assess the cytotoxic potential of GLU for the first time in prostate cancer (PC) cells representing different stages of the tumor. Furthermore, this study examined the potential synergistic activity of GLU in combination with docetaxel (DOC).Methods. Two different cell lines were used, LNCaP and PC-3. Concentration-response curves were assessed. The tested groups per cell line were, control, GLU, DOC and combination. Treatment duration was 72 h. Cytotoxicity was assessed using sulforhodamine B (SRB) assay and half maximal inhibitory concentration (IC50) was calculated. Synergy analyses were performed using Calcusyn®software. Subsequent mechanistic studies included β-glucosidase activity assay, glucose uptake and apoptosis studies, namely annexin V-FITC assay and the protein expression of mitochondrial pathway signals including Bcl-2, Bax, Caspase 9 and 3 were assessed. Data are presented as mean ± SD; comparisons were carried out using one way analysis of variance (ANOVA) followed by Tukey-Kramer’s test for post hoc analysis.Results. GLU induced cytotoxicity in both cell lines in a concentration-dependent manner. The IC50 in PC-3 cells was significantly lower by 19% when compared to that of LNCaP cells. The IC50 of combining both drugs showed comparable effect to DOC in PC-3 but was tremendously lowered by 49% compared to the same group in LNCaP cell line. β-glucosidase activity was higher in LNCaP by about 67% compared to that determined in PC-3 cells while the glucose uptake in PC-3 cells was almost 2 folds that found in LNCaP cells. These results were directly correlated to the efficacy of GLU in each cell line. Treatment of PC cells with GLU as single agent or in combination with DOC induced significantly higher apoptosis as evidenced by Annexin V-staining. Apoptosis was significantly increased in combination group by 4.9 folds and by 2.1 Folds when compared to control in LNCaP cells and PC-3 cells; respectively. Similarly, the expression of Bcl-2 was significantly decreased while Bax, caspase 9 and 3 were significantly increased in the combined treatment groups compared to the control.Conclusion. GLU has a synergistic effect in combination with DOC as it increases the cell kill which can be attributed at least partially to apoptosis in both the tested cell lines and it is suggested as a new combination regimen to be considered in the treatment of the prostate cancer. Further experiments and clinical investigations are needed for assessment of that regimen. Prostate cancer (PC) is among the most frequently diagnosed solid tumors in men aside from skin cancer. PC is the second leading cause of cancer death in American men, behind only lung cancer. The public health burden of prostate cancer is outspread. A total of 180,890 new cases of prostate cancer and 26,120 deaths from the disease are anticipated in the United States in 2016. Being old, African man with a family member with PC are all considered risk factors for PC , 1993. Sβ-glucosidase activity and glucose uptake in each cell line is also investigated.DOC is a semi-synthetic taxane analogue from the European yew (Taxus baccata) , 2004. DGLU was obtained from Threshold Pharmaceuticals . DOC was purchased from LC labs . Sulforhodamine B sodium salt (SRB) dye was purchased from Sigma-Aldrich . Cell culture media, fetal bovine serum (FBS), Penicillin/streptomycin solution, were obtained from Invitrogen .® HTB14™)  was also used. Cells were maintained as described previously . Human brain cancer cells U87 MG cell line  and each time with six replicates . The half maximal inhibitory concentration (IC50) was calculated using Graphpad Prism 5 software . Drug interactions were analyzed by Calcusyn program version 2.1  based on the analytical method of Chou and Talalay  according to the manufacturer’s instructions and the β glucosidase activity is calculated as described  as described previously , 2013.Western blotting was performed as described previously by P < 0.001. All statistical analysis was performed using Graph pad InStat software, version 3.05 .Data are presented as mean ± SD; comparisons were carried out using one way analysis of variance (ANOVA) followed by Tukey-Kramer’s test for post hoc analysis. Statistical significance was acceptable to a level of In order to investigate the effect of GLU, DOC and their combination, concentration–response curves of each drug as single agent were assessed and compared to those obtained from combining the two agents. SRB assay was performed as described before , 1990 anp < 0.001  for PC-3 and LNCaP cells, respectively , the high-dose of DOC as monotherapy is associated with significant toxic effects , 2001. FDrug combination is most widely used in treating the most dreadful diseases, such as cancer and AIDS. The main aims are to achieve synergistic therapeutic effect, to fulfill dose and toxicity reduction, and to minimize or delay the induction of drug resistance , 2010. Rin vivo experiments performed on intact human brain tumors demonstrated that their metabolism involves extensive glucose oxidation (β-glucosidase activity. Interestingly, it was found that β-glucosidase activity in LNCaP cells is 67% significantly higher than that in PC-3 cells, with 49% decrease and 68% decrease compared to U87 MG in LNCaP and PC-3; respectively.These cytotoxicity results raised the need for more investigations. Therefore, we assessed the difference in the uptake and hydrolysis of the GLU in both cell lines. Stage IV glioblastoma multiform U87 MG cells are known for their elevated glycolysis and glucose uptake , 2009. Mxidation , 2012 anxidation , 2012. Tβ-glucosidase activity in this breast cancer cell line. Additionally, β-glucosidase enzymatic activities in all cell lines. GLU has been identified as a substrate for cytosolic and lysosomal β-glucosidases (This observation was contradictory to the IC50 values computed for both cell lines let for other previously reported data on GLU. osidases , 1996 thβ-glucosidase levels seem only to be effective when transport proteins are expressed as well, may lend support to this view (β-glucosidase had a negligible effect (U87 MG cells significantly showed the highest glucose uptake, while glucose uptake was found to be significantly higher in PC-3 cells by nearly 2 folds compared to LNCaP cells, with 33% decrease and 65% decrease compared to U87 MG cells in PC-3 and LNCaP cells; respectively. The higher glucose uptake in PC-3 is in-line with previous data indicating that glycolysis is higher in aggressive tumors , 2012. This view , 1999. Ce effect , 2006.The combination of GLU/DOC resulted in a promising cytotoxicity in PC. Therefore, the current study was further substantiated in order to investigate the underlying mechanisms. Since induction of apoptosis is an important mechanism of cytotoxicity of both DOC , 2005 anBcl-2 family members are important in the regulation and control of the intrinsic apoptosis pathway. In the current study, the expression of Bcl-2 was significantly decreased on treatment with the combination compared to the control in both tested cell lines. DOC was reported to induce down-regulation of Bcl-2 in C4-2B PC cell line , 2008 anThe subsequent step was testing the effect of the different treatments on caspase-9 and caspase-3. Caspase-9 is the essential initiator caspase required for apoptosis signaling through the mitochondrial pathway and is activated on the apoptosome complex , 2012. Cin vitro results are extrapolated from bench to the clinical setting.In the current study, PC-3 cells were less sensitive to the drug than LNCaP. Similar finding was recently reported , 2014. Nin vitro data collected from the current study, when well analyzed, would point out, for the first time, to the oncolytic activity of GLU in two prostate cell lines; namely androgen-dependent LNCaP and androgen-independent PC-3 cells. Glucose uptake was found to be more rate limiting factor for GLU potency in PC cells than β-glucosidase. In addition, both drugs have shown synergistic apoptotic effects when combined together through completely diverse mechanisms of action. Finally, the potential merit of GLU/DOC combination warrants further investigations.In conclusion, based on these broad observations, one could conclude that the 10.7717/peerj.2168/supp-1Supplemental Information 1Click here for additional data file.


Malaria and HIV infections are both highly prevalent in sub-Saharan Africa, with HIV-infected patients being at higher risks of acquiring malaria. The majority of antiretroviral (ART) and anti-malarial drugs are metabolized by the CYP450 system, creating a chance of drug-drug interaction upon co-administration. Limited data are available on the effectiveness of the artemether-lumefantrine combination (AL) when co-administered with non-nucleoside reverse transcriptase inhibitors (NNRTIs). The aim of this study was to compare anti-malarial treatment responses between HIV-1 infected patients on either nevirapine- or efavirenz-based treatment and those not yet on ART (control-arm) with uncomplicated falciparum malaria, treated with AL.This was a prospective, non-randomized, open-label study conducted in Bagamoyo district, with three arms of HIV-infected adults: efavirenz-based treatment arm (EFV-arm) n = 66, nevirapine-based treatment arm (NVP-arm) n = 128, and control-arm n = 75, with uncomplicated malaria. All patients were treated with AL and followed up for 28 days. The primary outcome measure was an adequate clinical and parasitological response (ACPR) after treatment with AL by day 28.Day 28 ACPR was 97.6%, 82.5% and 94.5% for the NVP-arm, EFV-arm and control-arm, respectively. No early treatment or late parasitological failure was reported. The cumulative risk of recurrent parasitaemia was >19-fold higher in the EFV-arm than in the control-arm . The cumulative risk of recurrent parasitaemia in the NVP-arm was not significantly higher than in the control-arm . The median (IQR) day 7 plasma concentrations of lumefantrine for the three arms were: 1,125 ng/m (638.8-1913), 300.4 ng/ml (220.8-343.1) and 970 ng/ml (562.1-1729) for the NVP-arm, the EFV-arm and the control-arm, respectively (P < 0.001). In all three arms, the reported adverse events were mostly mild.After 28 days of follow-up, AL was statistically safe and effective in the treatment of uncomplicated malaria in the NVP-arm. The results of this study also provide an indication of the possible impact of EFV on the performance of AL and the likelihood of it affecting uncomplicated falciparum malaria treatment outcome. Malaria and HIV-1 are the most common infections in sub-Saharan Africa and to a lesser extent in other developing countries ,3. WorldThe World Health Organization (WHO) recommends the use of artemisinin-based combination therapy (ACT) as first-line treatment for uncomplicated falciparum malaria in all malaria-endemic countries . ArtemetArtemether is metabolized to dihydroartemisinin (DHA) via cytochrome P450 (CYP) CYP3A4, CYP2B6 and possibly CYP2A6 . LumefanThe treatment guidelines for uncomplicated malaria set by the National Malaria Control Programmes in sub-Saharan Africa do not discriminate between the dosing regimen of AL in non-HIV infected patients and those undergoing ART.To date, most of the reported studies describe the effect of HIV-1 infection on malaria treatment outcome in the absence of antiretroviral drug treatment -7,12-20.This study was conducted between May 2010 and August 2012 at an HIV clinic at Bagamoyo District Hospital. Bagamoyo district is an area of moderate malaria transmission. In this district, the peak time for malaria transmission peak time is usually around April to August. The study was approved by the Muhimbili University of Health and Allied Sciences (MUHAS) research and ethics committee and was conducted according to Good Clinical Practice. To ensure confidentiality, patients were identified by special identification numbers.This study involved HIV-1 infected adults receiving ART as 200 mg NVP twice daily or 600 mg EFV at night for more than two months and those not yet on ART. Enrolled in the study were 128 patients in the nevirapine-based treatment arm (NVP-arm), 66 patients in the efavirenz-based treatment arm (EFV-arm) and 75 patients not yet on ART (control-arm).Plasmodium falciparum with no signs of complicated (severe) malaria; no history of an allergic reaction or serious side effects to AL or treatment with anti-malarial drugs for at least four weeks prior to enrollment; no evidence of chronic diseases, such as renal or liver failure; not on anti-tuberculosis drugs for at least three months prior to enrollment; not pregnant or a nursing mother; easy accessibility to the health-care facility (travel time < 2 hours) and willingness to attend for the stipulated follow-up visits. Before enrollment, written informed consent was obtained from all patients. All patient information was recorded in a case report form (CRF). The enrolled patients were encouraged to take their ART and AL as prescribed.All HIV-1 infected patients presenting at the HIV clinic for routine medical care, with fever and other symptoms suggestive of uncomplicated falciparum malaria such as chills, sweats, headaches, muscle aches, nausea, vomiting, diarrhoea, body weakness, poor appetite, pallor and enlarged spleen were screened for eligibility. Patients were only enrolled into the study after meeting the inclusion criteria: Aged ≥ 18 years; reported fever within the last 24 hours and/or an axillary temperature ≥ 37.5°C and with any of the above-mentioned symptoms of uncomplicated falciparum malaria; haemoglobin ≥ 7 g/dl and ≥ 35 kg body weight; microscopically-confirmed This was a prospective, non-randomized, open-label, parallel and three-arm study. Patients were followed up for 28 days. Patients meeting the inclusion criteria were enrolled and took the full dose (three-day course) of AL  at 0, 8, 24, 36, 48 and 60 hrs. The first and fifth doses of AL were taken by direct observed therapy (DOT) with 250 ml of milk (3.5% fat). The other four doses were taken at home. All patients were given verbal instructions on dosing intervals and on the importance of combining treatment with fatty meals. Additionally, patients were supplied with 10 extra 250 ml packets of milk (3.5% fat) to be taken with the rest of the doses at home. For the first and fifth doses, which were given by DOT and paracetamol was administered to all febrile patients.Patients involved in this study were counseled to abstain from using alcohol, tea, caffeine and any drugs which may induce CYP3A4, such as griseofulvin, prednisolone, phenytoin, carbamezapine and phernobarbital. Non-prescription drugs, herbal medicines, oral contraceptives, grapefruits or grapefruit juice were also prohibited during the study.At enrollment all patients gave a finger-prick blood sample for thick smear and for haemoglobin (Hb) estimation. Blood slides for malaria parasites were all read at Ifakara Health Institute-Bagamoyo Research and Training Centre (IHI-BRTC). Venous blood was collected at pre-determined times for quantification of lumefantrine plasma concentrations. Patients’ baseline CD4+ cell count was obtained from their records, the timeline being within 3 months prior to study enrollment.Laboratory and clinical assessments were conducted on days 2, 3, 7, 14, 21 and 28 or on any day of recurrent illness. A reminder was sent to all patients by a study nurse via telephone about their medication and study visit schedules. Patients were encouraged to return to the study site any time they felt ill. Patients who failed to return on the scheduled day were visited and assessed at home. If the study nurse failed to locate a patient’s house, they were classified as lost to follow-up. Any additional medications taken during the study period were documented in the CRF.The time to recurrent parasitaemia or the risk of recurrent parasitaemia (RP) was defined as the number of days between taking the first dose of AL and the day of microscopically detecting malaria parasites in the thick blood film. The time at risk ended whenever one of the following conditions occurred: RP, loss to follow-up, withdrawal, or end of follow-up period .P. falciparum during the 28 days of follow-up were treated with either quinine tablets or injection as described in the malaria treatment guidelines (2006) of Tanzania [Patients with microscopically-confirmed Tanzania .All thick blood smears were stained with 10% Giemsa stain for 30 minutes. Parasite density was estimated by counting the number of asexual parasites per 200  white blood cells (WBC) on a thick smear. Parasite density per μl was calculated by assuming a WBC count of 8,000 per μl . All thiBlood samples from patients (4 ml) were collected in heparinized vacutainer tubes and centrifuged (×2000 g for 10 min) immediately to obtain plasma. Aliquots of plasma were transferred into 1 ml cryo-tubes, and stored at −80°C at the Ifakara Health Institute–BRTC until transfer to MUHAS for analysis. All patients’ plasma samples were analyzed at the MUHAS-Sida bio-analytical laboratory in Dar es Salaam. Lumefantrine concentrations were quantified using an HPLC method with UV detection as previously reported . The coeAn adequate clinical and parasitological response in patients at 28 days after anti-malarial treatment was the primary study objective. The WHO guidelines for assessment and monitoring of anti-malarial drug efficacy for the treatment of uncomplicated falciparum malaria were used in the evaluation of the time to RP after treatment with AL . AccordiThis study was considered to be exploratory. In total, 269 HIV-1 infected adult patients with uncomplicated falciparum malaria were enrolled with a minimum sample size of at least 50 patients for each arm . The datA total of 1,528 patients presenting at the HIV clinic with fever and other symptoms suggestive of malaria infection were screened. Among the screened patients, 316 (21%) had positive thick blood smears for malaria parasite. In total 269 (85%) patients met the inclusion criteria and were enrolled .In the current study, about 48% of the enrolled patients had CD4 cell counts of < 350 cells/μl. Patients with high parasitaemia had low CD4 cell counts and there was a strong association between CD4 cell counts and parasitaemia (P < 0.001).No early treatment failure or late parasitological failure was observed in the three arms. Overall, after day 28 of follow-up, 97.6% , 82.5%  and 94.5%  of patients in the NVP-arm, EFV-arm and control-arm, respectively, had no recurrent parasitaemia, thus meeting the WHO criteria for ACPR. The differences in the treatment outcome in the three arms were highly statistically significant (P < 0.001)  patients had lumefantrine plasma concentrations measured on day 7 after initiation of treatment. The median (IQR) day 7 plasma concentrations of lumefantrine for the three arms were: 1,125 ng/ml (638.8-1913), 300.4 ng/ml (220.8-343.1) and 970 ng/ml (562.1-1729) for the NVP and EFV-based treatment arms and for the control-arm, respectively. The difference in day 7 lumefantrine plasma concentrations between the EFV-arm and the control-arm was statistically significant (P < 0.001), while there was no statistically significance difference in the day 7 lumefantrine concentration between the NVP-arm and the control-arm (P = 0.063). In all three arms, the median lumefantrine concentration was significantly lower in patients with RP as compared to those with no RP  as opposed to a subject with poor immunity. However, in patients with poor immunity, the initial rate of parasite clearance is determined by the intrinsic activity of the drug, the susceptibility of infecting parasites and the drug levels achieved . The patPossible DDIs between ACT and NNRTIs in HIV-1 patients without malaria have been reported in a few studies -34. ThisP. falciparum malaria for a first-line drug in non-HIV patients should be at least 90% and preferably >95% [P. falciparum treated with AL is >95% [In the present study, all patients from the three arms were treated with artemether-lumefantrine and followed up for 28 days to determine malaria treatment response (ACPR). Based on WHO recommendations, the cure rates for bly >95% . Various is >95% . Under t is >95% , this stThe observed results in the EFV-arm may be due to possible DDIs between EFV and lumefantrine during the course of treatment as a result of the induction of common metabolic enzyme CYP3A4, leading to increased clearance of lumefantrine and artemether -30,42. AThese results are in agreement with findings from previous studies, which indicated a possible malaria treatment failure in patients treated with AL and EFV ,45 and bLumefantrine absorption varies considerably among individuals with its bioavailability being improved by fatty meal intake. As this study was unsupervised, poor adherence to treatment or inadequate fatty meal intake might have contributed to the observed sub-optimal day 7 lumefantrine plasma concentrations in the three arms, thus increasing the risk of recurrent parasitaemia. However, an AL cure rate of >96% was recently reported irrespective of whether it was given under supervision, with food or unsupervised .Differences in immune status among the studied population might have also contributed to the observed differences in the ACPR between the three arms. Patients in the EFV-arm had lower mean CD4 cell counts compared with those in the NVP and control-arms, although the differences did not reach statistical significance (P = 0.629). This is in line with previous reports ,17. Studet al. reported high risks of recrudescence in patients with high BPC and low plasma concentration of lumefantrine [Malaria parasite density was slightly higher among patients in both the NVP-arm and the EFV-arm than in the control-arm, although this difference was not statistically significant. Lower CD4 cell counts in the EFV and NVP-arms than in the control-arm might also have contributed to higher parasite density. This finding is in agreement with other studies, which documented higher parasite density with decreased immunity among HIV-1 infected patients ,17,46,47fantrine .P. falciparum parasitaemia, while the presented fever was caused by opportunistic infections. Reports from other studies have indicated that the febrile illness that occurs more frequently in HIV/AIDS patients, especially those with low CD4 cell counts, even in the presence of malaria infection, may be caused not by malaria but by other opportunistic infections or may be due to adverse drug reactions, thus complicating the diagnosis of malaria [P. falciparum parasitaemia among ART-treated patients and healthy people, respectively [While the results presented and discussed above are of clinical importance in improving the quality of life for HIV-1 infected patients on ART with uncomplicated falciparum malaria and treated with AL, nevertheless, they are not without limitations. In the present study, the enrolled patients were carefully screened for fever history, the presence or absence of other obvious causes of fever and the researchers carefully followed up guidelines for malaria screening and diagnosis ,36, neve malaria ,52-54. Tectively ,56.P. falciparum infection is reported to be equal or superior to routine microscopy (but inferior to expert microscopy) and do not generate all information provided by microscopy [Decreasing the risk of false negative malaria, especially in patients with low malaria parasitaemia, using more sensitive diagnostic methods such as the malaria rapid diagnostic test (MRDT) and quantitative real-time polymerase chain reaction (PCR), could have complimented the thick blood smear film method. However, the MRDT and PCR are not without limitations as it is in the case of thick blood film. The accuracy of RDT for the diagnosis of uncomplicated croscopy . The thicroscopy ,59.P. falciparum deficiency (Pfhrp2 gene deletions) and due to cross-reactions between Plasmodium species. The sensitivity of MRDT has been reported to be reduced to < 75% with < 1000 parasites/μl [Thick blood films have been reported to give conflicting results in various studies -62. Howesites/μl ,64,65. Fsites/μl ,62,64,66sites/μl . This stsites/μl ,61,64,68In the present study, malaria parasites were not genotyped to differentiate recrudescence from re-infection ; in areaIn conclusion, the findings of this study suggest that AL is safe and effective in the treatment of uncomplicated falciparum malaria in patients receiving NVP-based ART compared to those receiving EFV-based ART. The results of this study also provide an indication of the possible impact of EFV on the performance of AL and the likelihood of it affecting malaria treatment outcome. Surveillance on the effectiveness and efficacy of AL in HIV infected patients, particularly those on EFV-based ART, should be performed in order to elucidate the role of the latter in the treatment outcome of uncomplicated falciparum malaria.The authors declare that they have no competing interests.OMSM and SP conceived the study, participated in the study design, coordination, data analysis and manuscript writing. BM participated in study design, data collection and analysis and in the preparation and writing of the manuscript. BN participated in the data analysis as well as manuscript preparation. All authors participated in reading and approving the final manuscript.


Few preclinical or clinical studies have evaluated the effect of anesthetics on motor evoked potentials (MEPs), either alone or in the presence of conditioning stimuli such as deep brain stimulation (DBS). In this study we evaluated the effects of two commonly used anesthetic agents, propofol and ketamine (KET), on MEPs elicited by intra-cortical microstimulation of the motor cortex in a rodent model with and without DBS of the dentatothalamocortical (DTC) pathway. The effects of propofol anesthesia on MEP amplitudes during DTC DBS were found to be highly dose dependent. Standard, but not high, dose propofol potentiated the facilitatory effects of 30 Hz DTC DBS on MEPs. This facilitation was sustained and phase-dependent indicating that, compared to high dose propofol, standard dose propofol has a beta-band excitatory effect on cortical networks. In contrast, KET anesthetic demonstrated a monotonic relationship with increasing frequencies of stimulation, such that the highest frequency of stimulation resulted in the greatest MEP amplitude. KET also showed phase dependency but less pronounced than standard dose propofol. The results underscore the importance of better understanding the complex effects of anesthetics on cortical networks and exogenous stimuli. Choice of anesthetic agents and dosing may significantly confound or even skew research outcomes, including experimentation in novel DBS indications and paradigms. Evoked and event-related potentials are frequently used in laboratory and human research as well as clinical practice, and anesthesia has substantial effects on them. It has been reported that motor cortical excitability during transcranial magnetic stimulation (TMS) depends on anesthetics. Intravenous propofol suppresses motor evoked potentials (MEPs) while intravenous ketamine (KET) does not suppress MEPs and may even facilitate MEPs  Vitek, . The denOur findings suggest that; (a) cortical excitability, as indexed by the amplitude of MEPs, was higher under KET than propofol; (b) MEP amplitude was higher during ON-DBS, compared to OFF-DBS in all experiments; (c) when combined with DTC DBS, KET was associated with increased MEP amplitude at high DBS frequencies; (d) there was an interaction between propofol dose and DBS frequency such that standard-dose propofol, but not higher dose propofol, potentiated the facilitatory effects of 30 Hz DBS on MEP amplitude and; (e) both standard-dose propofol and KET showed phase-dependency between ICMS and DBS pulses, indicating that these anesthetics may, in a dose-dependent fashion, induce oscillatory effects on thalamocortical networks.All experiments were performed using male Sprague Dawley rats . The animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved animal facility in a climate-controlled environment that included a 12-h light/dark cycle and free access to food and water. All procedures were performed under an experimental protocol approved by the Institutional Animal Care and Use Committee (IACUC) and complied with Public Health Service policy. The experimental model utilized ICMS as the test stimulus Figure . The conElectrodes were implanted under aseptic conditions as previously described  at 125% of motor threshold for forelimb activation. A random interval (± 500 ms) was added to the inter-stimulus interval to minimize the possibility for time-locking between the intracortical burst and the chronic cerebellar stimulation pulses. Each intracortical burst consisted of a train of three charge-balanced square-wave pulses with an intra-burst frequency of 330 pulses per second and a pulse width of 400 µs. Forearm EMG activity was time-locked to the onset of the intracortical burst using stimulus trigger signals. Throughout the experiments, heart rate was continuously recorded as a function of time.Upon completion, histological examination was conducted as previously described . Briefly, each MEP response between 8 ms and 17 ms after the first ICMS pulse was rectified and averaged. Then the average rectified EMG prior to stimulation was subtracted to obtain the corresponding MEP magnitude.p < 0.05 for these analyses.The effects of three different anesthetic conditions  irrespective of time in the OFF-DBS condition were tested with a one-way ANOVA. One-way ANOVA was also used to evaluate the effects of time duration of anesthesia on MEP amplitudes in the OFF-DBS condition for each anesthetic condition separately. The effects of ON-DBS and OFF-DBS under the three anesthetic conditions were analyzed using two-way ANOVA with repeated measures of median MEP values. Two-way ANOVA was also used to evaluate the interaction of the different anesthetic conditions over time on MEP amplitudes. Futhermore, the effects of various DBS frequencies on MEP amplitudes under different anesthetic conditions were analyzed with two-way ANOVA with repeated measures, using median MEPs for each parameter and animal. Statistical significance was accepted at A bootstrap method was adopted to further examine any potential effect of the relative timing  of the temporally preceding LCN DBS pulse to the delivery of the ICMS pulses on MEP magnitude Figure . A totalp = 0.0225, F = 4.46), and Bonferroni’s multiple comparison test showed that MEP under KET anesthesia was greater than that under PROP-h (p < 0.05), while there was no statistical significance among the other pairwise comparisons.Nine rats per anesthesia group, for a total of twenty-seven rats, underwent LCN DBS electrode implantation and MEP testing. During the OFF-DBS condition, the median MEP amplitude per animal under KET, PROP-s, and PROP-h was 47.5 ± 31.6 µV, 29.3 ± 11.0 µV and 20.4 ± 5.8 µV (mean ± s.d.), respectively. Anesthetics had a statistically significant effect on MEP during OFF-DBS condition  = 5.545) and DBS  = 25.89) but failed to identify an interaction between anesthesia and DBS (p = 0.4604). Post hoc analysis demonstrated a significant difference between the ON-DBS and OFF-DBS conditions for both KET (p < 0.01 with Bonferroni correction) and PROP-s (p < 0.05 with Bonferroni correction).A general result for this experiment is that, regardless of the pulse frequency of LCN DBS, MEPs were observed to be larger during ON-DBS conditions than in the OFF-DBS condition for both KET and PROP-s anesthetics Figure . This obp < 0.0001, F = 22.21), but no main effect for time  = 0.6681). Post hoc analysis revealed that MEPs under KET were significantly greater than PROP-h at 80–120 min (p < 0.05 with Bonferroni correction). No other significant differences were found between KET and PROP-s or PROP-s and PROP-h.To determine the effects of anesthetic agent and dose on cortical excitability over time in the absence of DBS, the amplitude of the MEPs recorded during consecutive OFF-DBS epochs were Figure . A two-wp = 0.96). The average median magnitudes of the MEPs recorded at the initial 10-min OFF-DBS epoch were 43.8 µV, 41.0 µV and 41.4 µV for the KET, PROP-s and PROP-h groups, respectively. Although MEP amplitude tended to decrease over time within both PROP-s  = 1.472) and PROP-h  = 1.846) groups, these changes were not significant. The mean reduction in MEP amplitude was 16.8 µV and 24.3 µV, respectively, for PROP-s and PROP-h, over a period of 240 min. In contrast, MEP amplitudes under KET increased over time initially, reaching a maximum of 60.9 µV at 120 min. However, the increase was not significantly different  = 0.325) compared to any time point over the 240 min period.We further evaluated each anesthetic individually to determine whether its effect on cortical excitability, in the absence of DBS, varied over time by comparing the amplitude of the MEPs recorded across each successive OFF-DBS epoch. Within each anesthetic conditions there were no significant differences in MEP amplitudes over time relative to the initial 10-min OFF-DBS epoch  = 17.483, p = 0.042), and the Greenhouse-Geisser correction (ε = 0.753) was used. Two-way ANOVA with repeated measures revealed a significant effect for anesthesia  = 5.135), DBS pulse frequency  = 5.539), and the interaction between anesthesia and frequency  = 2.687). In order to understand the relationship between the DBS pulse frequency and the evoked MEP, we performed a post hoc analysis independently for each anesthetic regimen comparing OFF-DBS to ON-DBS at 20, 30, 50 or 100 Hz stimulation . In contrast, PROP-s was associated with an increase in MEP amplitude that peaked at 30 Hz DBS but then returned to near baseline levels  at higher frequencies of stimulation . Finally, PROP-h was not associated with any significant frequent-dependent modulation of MEP magnitude.To determine the effect of propofol dose on frequency-specific ON-DBS modulation of MEPs, the amplitude of the MEPs recorded under KET, PROP-s and PROP-h were compared. In the two-way ANOVA with repeated measures, the Mauchly’s test of sphericity indicated that the assumption of the sphericity was violated  when DBS was set to 30 Hz, but not other frequencies. The findings could be consequent to the potentiation effects between the exogenous 30 Hz stimulation and propofol-induced beta-band paradoxical excitation, previously reported in humans by McCarthy et al.  see Sec. Under PLike PROP-s, we also found a LCN DBS frequency dependency of MEPs under KET and a phase association was also identified. This association occurred at 20 Hz  and 30 Hz  techniques during anesthesia (Ching et al., Our findings are congruous with previous human studies evaluating the effects of propofol or KET on TMS-elicited MEPs. A detailed review of the effects of various anesthetics on TMS-electited MEPs can be found in Ziemann’s review paper Ziemann, . PropofoIn summary, our findings stress the importance of understanding the potential effects of different anesthetic agents on experimental DBS and electrophysiological research. These findings can significantly impact the translation of DBS of the DTC pathway for post-stroke rehabilitation as well as other emerging DBS therapies and techniques.Regarding conflict of interest, authors Andre G. Machado and Kenneth B. Baker have potential financial conflict of interest with this research related to intellectual property and distribution rights from intellectual property in IntElect Medical and possible future distribution in Enspire, ATI and Cardionomics. The Cleveland Clinic COI committee has approved a plan for managing the conflict of interest in the conduct of this research. The authors have adhered to the management plan in the conduct and reporting of research findings. None of these entities had any role in the research or preparation of the manuscript. The research was funded by NIH grant R01HD061363.


There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is a principal molecule in the pathogenesis of Parkinson disease (PD). We previously reported that total A-syn levels in cerebrospinal fluid (CSF), measured with the specific enzyme-linked immunosorbent assay (ELISA) developed by ourselves, were decreased in patients with PD, and suggested the usefulness of A-syn in CSF and plasma as a biomarker for the diagnosis of PD. After our report, a considerable number of studies have investigated the levels A-syn in CSF and in blood, but have reported inconclusive results. Such discrepancies have often been attributed not only to the use of different antibodies in the ELISAs but also to interference from hemolysis. In this study we measured the levels of A-syn in CSF and plasma by using our own sandwich ELISA with or without heterophilic antibody (HA) inhibitor in 30 patients with PD and 58 age-matched controls. We thereby revealed that HA interfered with ELISA measurements of A-syn and are accordingly considered to be an important confounder in A-syn ELISAs. HA produced falsely exaggerated signals in A-syn ELISAs more prominently in plasma samples than in CSF samples. After elimination of HA interference, it was found that hemolysis did not have a significant effect on the signals obtained using our A-syn ELISA. Furthermore, plasma levels of A-syn were significantly lower in the PD group compared with the control group following elimination of HA interference with an HA inhibitor. Our results demonstrate that HA was a major confounder that should be controlled in A-syn ELISAs, and that plasma A-syn could be a useful biomarker for the diagnosis of PD if adequately quantified following elimination of HA interference. It is pathologically characterized by the presence of Lewy bodies and Lewy neuritis in the substantia nigra and several other subcortical regions . α-Synuct=4741)” –19. HAs t=4741)” , 18 (S1(t=4741)” , 19. HA t=4741)” . Howevert=4741)” . Therefot=4741)” . With reBlood-based tests are less invasive and more suitable for mass screening than CSF-based tests. In fact, a considerable number of reports assessing A-syn concentrations in blood have been published to date , 21–27. http://www.wma.net/en/30publications/10policies/b3/index.html). The patients were registered in this institute from April 2009 to August 2013. In Study I, we compared the levels of A-syn between samples treated with and without HAI using the CSF or plasma samples. In Study II we quantified A-syn levels in CSF and plasma samples with HAI. In Study III, we quantified the levels of Hb in the CSF and plasma samples. In study II, we enrolled 30 patients with non-familial PD diagnosed according to the UK PD Society Brain Bank criteria ; 65.7 ± 9.7 yr) in the Department of Neurology, Kyoto prefectural University of Medicine. We also enrolled 58 participants as age-matched controls  in the same registration . Immediately after collection, the samples were cleared by centrifugation at 400 x g for 10 min at 4°C, and then a cocktail of protease inhibitors  was added to each sample. Blood was drawn through venipuncture into EDTA-containing collecting tubes soon after CSF collection, and plasma was separated by centrifugation at 2000 x g for 10 min at 4°C. CSF and plasma samples were aliquoted into polypropylene tubes, and stored at -80°C until used for ELISA.CSF samples were collected in polypropylene vials from PD and control cases in the morning (from 9 a.m. to 12 a.m.) through a lumbar puncture at the L3/L4 or L4/L5 interspace by referring to the Biologics Manual of the Parkinson’s Progression Markers Initiative biologics manual , pH 9.6, containing 0.02% (w/v) sodium azide, washed four times with PBST  containing 0.05% Tween 20) and incubated with 200 μl/well of blocking buffer (PBS containing 2.5% gelatin and 0.05% Tween 20) for 2 hours. After washing with PBST, 100 μl of the samples diluted with or without HAI  were added to each well and incubated at 37°C for 3 hours. Captured A-syn was detected using 0.2 μg/ml of FL-140 antibody (100 μl/well) diluted to 1:1000 in blocking buffer, followed by incubation with 100 μl/well  of HRP-labeled anti-rabbit antibody . Bound HRP activity was assayed by chemiluminescence using an enhanced chemiluminescent substrate . Chemiluminescence in relative light units was measured with a microplate luminometer . The standard curve for the ELISA was carried out in each plate using 100 μl/well of recombinant human A-syn  solution at different protein concentrations in PBS. Relative concentration estimates of total A-syn in the samples were calculated according to the standard curve obtained in each plate. To eliminate inter-assay variability as a confounding factor, all measurements were conducted in triplicate (unless otherwise noted) and performed using the same lot of standards. Furthermore, we placed internal control samples in each plate to adjust plate-to-plate variability. The intra-assay and inter-assay variance was less than 10%.Total A-syn levels in CSF and plasma were measured using a sandwich ELISA system 211-FL140 ELISA) as previously described with some modification [0 ELISA aThe hemoglobin (Hb) levels in CSF and plasma samples were measured using a Human hemoglobin ELISA Quantitation Kit from Bethyl Lab Inc  according to the manufacturer’s instructions.Blue native-PAGE was performed using Novex 4–12% gradient gels . The primary antibodies (C211 and FL-140) were prepared by addition of 5% Coomassie G-250 additive. Blue Native-polyacrylamide gels were then run at 150 V for 2 hours according to the manufacturer’s protocol, and the separated proteins were then transferred onto PVDF membranes . The membranes were then blocked with 5% nonfat milk in PBS containing 0.1% (v/v) Tween 20 for 30 min at RT, and subsequently incubated for 24 hours with plasma samples diluted to 50% in PBS, which exhibited either high or low HA activity in preparatory experiments. After washing with PBST, the membranes were incubated in HRP-labeled anti-human immunoglobulin antibody  for 3 hours. Immunosignals were visualized using chemiluminescence .Mann-Whitney's U tests were used for comparisons between two independent groups. Correlation analysis was conducted using Spearman’s rank correlation coefficient test. The level of significance was set at p< 0.05. All analyses were performed with Graph Pad Prism for Windows .It is expected that optimal HAI concentration ranges will be specific to individual ELISA assay systems. To determine the appropriate HAI concentration for our A-syn ELISA (211-FL140 ELISA), we first performed an ELISA for recombinant A-syn (known concentration) diluted with various amounts of HAI. We found that use of high concentrations of HAI disrupted the ELISA performance , therefoWe used 68 paired samples of CSF and plasma obtained from 23 patients with PD and 45 controls to determine whether pretreatment with HAI could alter signals in the 211-FL140 ELISA . The other two samples, where plasma A-syn levels were slightly increased with HAI pretreatment, had the lowest plasma A-syn levels in this cohort measured in the absence of HAI .We conducted blue native-PAGE to confirm that the signal reduction observed in the plasma samples was caused by HA. The antibodies employed in our A-syn ELISA (211 and FL140) were separated by native PAGE and analyzed with immunoblotting. We then performed immunoblotting using the plasma samples as the primary antibodies and anti-human immunoglobulin as the secondary antibody to detect HA against both the capture (211) and detection (FL140) antibodies of our ELISA. We chose two plasma samples, with either high or low HA activity in the experiments of 211-FL140 ELISAs with or without HAI. In the plasma sample with high HA activity, there were strong bands that reacted with the FL-140 and 211 antibodies . ConversWe measured the concentrations of A-syn in 88 CSF and plasma  samples using the 211-FL140 ELISA with HAI pretreatment  and control groups (p = 0.10). Furthermore, the age of the PD patients and plasma A-syn levels were significantly correlated . In CSF We measured the levels of Hb as a direct hemolytic marker in the samples , and examined the correlation between the levels of Hb and A-syn measured using the 211-FL140 ELISA with or without the HAI pretreatment  and detection (FL140) antibodies employed in the A-syn ELISA. The HAI we used could prevent these immunoglobulins from binding the capture and reporter antibodies, despite detailed constituents of the HAI were not informed from the manufacture. These results suggest the following two hypotheses: 1) A-syn values greater than 50 ng/ml measured with the 211-FL140 A-syn ELISA without HAI pretreatment are likely to be falsely exaggerated by the presence of HAs in the examined samples; 2) HA interference is more prominent in plasma than in CSF. There have been two studies describing HA interference in Aβ ELISA. They found that HA generally affects micro-quantitative ELISA more strongly in plasma than in CSF, and produces false-positive rather than false-negative signals , 29. OurSecond, we also found that plasma A-syn levels with HAI pretreatment were significantly lower in the PD group than in the control group. Previous studies that quantified A-syn in plasma to elucidate its usefulness as a blood-based biomarker for the diagnosis of PD have lacked reproducibility , 21–27, Third, we did not find a significant relationship between the levels of Hb and CSF or plasma A-syn. In contrast, previous studies have reported that hemolysis is a confounding factor that provides a strong positive signal in A-syn ELISAs , 34, becPlasma A-syn was slightly higher than CSF A-syn, even with elimination of HA interference in this study. This result is in agreement with the report of Mollenhauer et al. , despiteWe found that the levels of CSF A-syn were positively correlated with the levels of plasma A-syn in the PD group, but not in the control group. This observed difference between the PD and control groups is likely due to the presence of subjects in the PD group, but not the control group, who were characterized by lower A-syn levels in both CSF and plasma . BecauseIn conclusion, the present study indicates that the presence of HA is a major confounder in some ELISAs, including our A-syn ELISA. HA interference was more prominent in plasma than in CSF. Upon elimination of HA interference in the plasma, plasma A-syn levels were significantly lower in the PD group than in the control group. Moreover, after HA interference in the plasma was eliminated the plasma A-syn levels significantly correlated with the CSF A-syn levels in the PD group. These results indicate that plasma A-syn could be useful as a blood-based biomarker for the diagnosis of PD when adequately quantified by eliminating the interference of HAs.S1 Fig(A) Schema of how ELISAs detect the antigen (Ag), which is trapped between capture and reporter antibodies (Ab). (B) The form of HA interference in ELISAs which leads to a false positive result. The HA binds to both the capture and the reporter antibody simulating the presence of Ag in its absence. (C) The form of HA interference in ELISAs which leads to a false negative result. The HA binds to the capture (or the reporter) antibody and prevents antigen-antibody interaction.(TIF)Click here for additional data file.S2 Fig Scatter plots of the age of the subjects versus CSF A-syn levels in the control (A) and PD (B) groups.  Scatter plots of the age of the subjects versus plasma A-syn levels in the control (C) and PD (D) groups. The lines in the graphs represent regression lines, with solid and dashed lines representing significant and non-significant correlations, respectively. Regression analyses revealed a non-significant correlation between the age of the controls and CSF A-syn levels  or plasma A-syn levels , as well as between the age of the PD patients and CSF A-syn levels . The correlation between the age of the PD patients and the levels of plasma A-syn was significant .(TIF)Click here for additional data file.


Thrombopoietin (TPO), a growth factor primarily involved in thrombopoiesis may also have a role in the pathophysiology of sepsis. In patients with sepsis, indeed, TPO levels are markedly increased, with disease severity being the major independent determinant of TPO concentrations. Moreover, TPO increases and correlates with ex vivo indices of platelet activation in patients with burn injury upon sepsis development, and may contribute to depress cardiac contractility in septic shock. Still, the role of TPO in sepsis pathophysiology remains controversial, given the protective role of TPO in other experimental disease models, for instance in doxorubicin-induced cardiotoxicity and myocardial ischemia/reperfusion injury. The aim of our study was to define the contribution of TPO in the development of organ damage induced by endotoxemia or sepsis, and to investigate the effects of inhibiting TPO in these conditions.We synthesized a chimeric protein able to inhibit TPO, mTPOR-MBP, and studied its effect in two murine experimental models, acute endotoxemia and cecal ligation and puncture (CLP) model.P<0.01), and from 265.00±26.02 pg/mL to 373.70±26.20 pg/mL in the CLP model (P<0.05), respectively. Paralleling TPO levels, also platelet-monocyte aggregates increased, from 32.86±2.48% to 46.13±1.39% at 3 hours in the LPS model (P<0.01), and from 43.68±1.69% to 56.52±4.66% in the CLP model (P<0.05). Blockade of TPO by mTPOR-MBP administration reduced histological damage in target organs, namely lung, liver, and gut. In particular, neutrophil infiltration and lung septal thickening were reduced from a score of 1.86±0.34 to 0.60±0.27 (P<0.01) and from 1.43±0.37 to 0.40±0.16 (P<0.05), respectively, in the LPS model at 3 hours, and from a score of 1.75±0.37 to 0.38±0.18 (P<0.01) and from 1.25±0.31 to 0.13±0.13 (P<0.001), respectively, in the CLP model. Similarly, the number of hepatic microabscesses was decreased from 14.14±1.41 to 3.64±0.56 in the LPS model at 3 hours (P<0.001), and from 1.71±0.29 to 0.13±0.13 in the CLP model (P<0.001). Finally, the diameter of intestinal villi decreased from 90.69±3.95 μm to 70.74±3.60 μm in the LPS model at 3 hours (P<0.01), and from 74.29±4.29 μm to 57.50±1.89 μm in the CLP model (P<0.01). This protective effect was associated with the blunting of the increase in platelet-monocyte adhesion, and, on the contrary, with increased platelet-neutrophil aggregates in the circulation, which may be related to decreased neutrophil sequestration into the inflamed tissues. Conversely, circulating cytokine levels were not significantly changed, in both models, by mTPOR-MBP administration.In both models, TPO levels markedly increased, from 289.80±27.87 pg/mL to 465.60±45.92 pg/mL at 3 hours in the LPS model (Our results demonstrate that TPO participates in the development of organ damage induced by experimental endotoxemia or polymicrobial sepsis via a mechanism involving increased platelet-leukocyte adhesion, but not cytokine release, and suggest that blocking TPO may be useful in preventing organ damage in patients affected by systemic inflammatory response or sepsis. Thrombopoietin (TPO), a growth factor primarily involved in regulating thrombopoiesis , 2, has TPO is constitutively produced by liver and kidneys, and cleared from the circulation by platelets upon binding to the TPO receptor c-Mpl, followed by internalization of the bound ligand , 2. Its Several studies have previously reported that TPO levels are increased after endotoxin infusion and in patients with sepsis , especiaIn this study, we aimed to define the role of TPO in the development of organ damage induced by experimental endotoxemia or polymicrobial sepsis, and to investigate the effects of inhibiting TPO in these conditions. To this end, we generated and characterized for its inhibiting activity against TPO a soluble recombinant chimeric protein, mTPOR-MBP, composed of the extracellular portion of mouse TPO receptor mTPOR/cMpl fused to the maltose-binding protein (MBP) in order to increase its solubility. We then studied whether TPO inhibition by mTPOR-MBP may reduce organ damage in two widely used experimental models, one mimicking a systemic inflammatory response syndrome, induced by i.p. injection of lipopolysaccharides (LPS), the other a model of abdominal infection leading to polymicrobial sepsis, represented by the cecal ligation and puncture (CLP) model.malE gene encoding MBP. Escherichia coli strain BL21(DE3)pLysS cells  was transformed with the vector. Protein expression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 h at 37°C. Thereafter, cells were collected by centrifugation, frozen at -20°C, then thawed at 37°C, and lysed by sonication. The soluble fraction containing mTPOR-MBP fusion protein was loaded on a pre-packed amylose affinity column (New England Biolabs), and elution was performed with the same buffer supplemented with 10 mM maltose. mTPOR-MBP was finally dialysed against phosphate-buffered saline (PBS) and stored at -70°C.We generated mTPOR-MBP by fusing the extracellular portion of mouse TPO receptor mTPOR/c-Mpl to maltose-binding protein (MBP). The DNA fragment encoding murine c-Mpl-extracellular domain (26–491) was amplified by PCR through high-fidelity DNA polymerase , using 300 ng of murine c-Mpl cDNA  as a template. The following primers were used: Forward 5’CCGGAATTCATGAGCCAAGATGTCTTCTTGCTGGCCT3’; Reverse 5’TGCTCTAGATTACAGGAGCAGAGCAGTCAC3’, carrying, at the 5’-end, the sequence for EcoRI and XbaI restriction enzymes, respectively. The amplification product was first digested with EcoRI and XbaI and purified on agarose gel. The 1425 bp PCR product was then sub-cloned into the expression vector pMal-c2 digested with EcoRI and XbaI , downstream and in frame with the Protein was analysed by SDS-PAGE on 8% polyacrylamide gel under reducing conditions, and transferred onto nitrocellulose. The membrane was blocked with 5% BSA in Tris-buffered solution containing Tween-20 (TBS-T) overnight at 4°C, followed by incubation with either anti-murine c-Mpl monoclonal antibody , or anti-MBP rabbit polyclonal serum. Blots were probed with peroxidase-conjugated sheep anti-mouse , or goat anti-rabbit  antibodies, as appropriate, and developed with chemiluminescence reagents .Binding specificity of mTPOR-MBP with TPO was analysed by dot-blot as previously described . Brieflyin vitro was tested by using a short-term proliferative assay on the megakaryoblast cell line M-07 [The ability of mTPOR-MBP to block TPO biological activity ine M-07  and platine M-07 .5 M-07 cells were plated in a 96-wells microplate and stimulated with rTPO (50–1000 pg/ml), in the presence or absence of mTPOR-MBP (1–20 μg/ml) for 48 hours. Afterwards each well was pulsed with 1 μC of tritiated thymidine (Amersham) allowing incorporation for 16 hours. Cells were then harvested, and thymidine incorporation was determined in a scintillation counter after addition to each vial of 1mL of scintillation liquid.For the proliferative assay, 2x10For platelet aggregation experiments, blood was collected by clean venipuncture using a 19-gauge butterfly infusion set, without venous stasis, from healthy adult donors, who had not taken any medications for at least 2 weeks. Nine volumes of blood were withdrawn in 1 vol of 3.8% trisodium citrate. PRP was prepared by centrifugation for 15 minutes at 180g. Platelet poor plasma (PPP) was obtained by centrifugation at 2,000g for 10 minutes. Platelet aggregation in PRP was evaluated according to the Born's method , at 37°CIn preliminary experiments, mice were injected i.p. with 40 μg of mTPOR-MBP, and, for the next 24 hours, the presence of mTPOR-MBP in plasma was checked. Briefly, blood was drawn by the tail vein, centrifuged at 600g for 10 minutes in order to obtain plasma, and the presence of mTPOR-MBP was detected by western blot analysis, using the anti-MBP rabbit polyclonal serum.Three month-old male C57BL/6 mice were used for all experiments (the exact n/group distributions are specified in the figure legends). Mice were kept together in groups of five per cage, housed on a 12 h light-dark diurnal cycle with controlled temperature (21°C to 23°C) and provided with standard rodent diet and water ad libitum.Acute endotoxemia was induced by i.p. injection with 40 mg/kg of LPS from E. coli 0111:B4 (Sigma-Aldrich) in a volume of 250 μl sterile saline solution [To confirm the results obtained with LPS injection, we used a second model of polymicrobial sepsis induced by cecal ligation and puncture (CLP) . Mice weThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as institutional guidelines and Italian government regulations. The protocol was approved by the Institutional Review Committee for Animal Care and Use at the University of Turin (Protocol Number: 0008619-P). All surgery was performed under anesthesia, and all efforts were made to minimize suffering. In particular, buprenorphine (0.05 mg/kg i.p.) was administered before surgery as well as 6 hours after surgery to reduce postoperative pain, and animals were inspected for signs of illness and/or unusual behavior by research staff at least once per day. Whenever an animal's condition deteriorated  followed by the appearance of signs of imminent death  mice were killed by using deep inhalation anesthesia (isoflurane) followed by an overdose of barbiturate . The percentage of premature deaths in both models did not exceed 10%.2O. Portions of the formalin-distended lungs were harvested and used for histological analysis.At the indicated times , mice were anesthetized with tiletamine (80 mg/kg), and blood samples were collected from the inferior cava vein into tubes containing trisodium citrate (0.11 mol/liter). The examined time-points have been chosen on the basis of preliminary experiments performed in our laboratory, and of similar studies already published in the literature , 27, 28.Histological damage was evaluated on H&E-stained 5 μm paraffin sections by an expert pathologist (P.D.G.) blind to the sample identity. To measure lung injury, neutrophil infiltration and alveolar-capillary membrane thickening were scored by assigning a subjective value from zero (absent) to three (extensive) scale. In each tissue sample, five random fields were scored, and the mean value was calculated.To evaluate liver injury, the number of leukocyte microabscesses, i.e. discrete neutrophil infiltrates around the portal veins that derive from neutrophil infiltration within the liver parenchyma , was couGut injury was evaluated by measuring intestinal villus thickness in 5 randomly-selected fields.TPO levels in plasma were determined by ELISA (R&D Systems Inc.) according to the manufacturer’s recommendations, with the exception of a 3-fold plasma dilution.Cytokine  concentrations were measured in plasma samples using a fluorescent bead–based multiplex immunoassay , as previously described , 30.Leukocyte-platelet aggregates ex vivo were analyzed by two-colour staining of whole blood samples. Briefly, blood was collected, diluted 1:1 with Tyrodes’s Hepes-buffered saline (pH 7.4), and incubated 15 minutes at room temperature with a mixture of PE-Cy5-conjugated anti-mouse-CD45 monoclonal antibody , which binds all leukocytes, and FITC-conjugated anti-mouse-CD41  monoclonal antibody, which identifies platelets. Cells were then fixed with 1% paraformaldehyde and erythrocytes were removed by hypotonic lysis. Samples were washed twice with PBS-BSA 0.1% and resuspended in 0.5 ml PBS. Samples were analyzed on a EPICS–XL flow cytometer . Total leukocytes were identified by their positive staining with anti-CD45 in the CD45 versus side scatter dot plot, whereas lymphocyte, neutrophil and monocyte populations were discriminated by size and granularity in the forward scatter versus side scatter dot plot using a logic gate. The percentage of monocyte-platelet or neutrophil-platelet aggregates, therefore co-expressing both markers CD45 and CD41 over the total number of cells counted within the logic gates previously selected was calculated and used as an index of platelet-monocyte or platelet-neutrophil adhesion.t test, where appropriate. Relationship between variables was investigated by Spearman rank correlation coefficient.Data are expressed as mean ± S.E.M. Comparisons between groups were analyzed by ANOVA followed by Newman-Keul’s multicomparison test or by Student’s P value < 0.05 was considered significant.A All statistics were done with GraphPad Prism 4.00 for Windows .Aiming to block TPO actions in vivo, we designed and synthesized a chimeric protein, mTPOR-MBP, originated by the fusion of the extracellular portion of mTPOR/c-Mpl and of MBP, used to enhance its solubility. The soluble form of mTPOR-MBP was purified by affinity chromatography using an amylose column, and recognized by antibodies directed against MBP and TPOR/c-Mpl by western blot. mTPOR-MBP showed dose-dependent high-affinity binding for rTPO in dot-blot assay, whereas it did not recognized TNF-α and IL-1β . In addiPrevious studies have shown that TPO levels are increased after endotoxin infusion and in patients with sepsis , 15. HerIt is known that sepsis mortality is strictly related to the development of injury and failure in target organs . In bothThe effect of mTPOR-MBP on the development of organ damage induced by LPS administration was evaluated at 3 and 6 hours Figs . Lung inMoreover, mTPOR-MBP reduced the number of hepatic micro-abscesses around the centrolobular veins, consequent to massive infiltration of inflammatory cells into the hepatic parenchyma, at both 3 and 6 hours from LPS injection .In severe sepsis pathophysiology, intense pro-inflammatory reactions, which includes the secretion and release of several cytokines in blood, are usually associated with the development of collateral tissue damage and consequent organ failure . As expemTPOR-MBP alone did not show any effect on cytokine levels (not shown).Sepsis development has been associated with the appearance in the circulation of increased platelet-monocyte and platelet-neutrophil aggregates, which are believed to have a relevant role in the progression to the most severe forms of the disease . In partP<0.05), and between TPO levels and platelet-neutrophil adhesion  measured ex vivo. mTPOR-MBP administration significantly blunted the increase in both platelet-monocyte and platelet-neutrophil adhesion observed at 3 hours from LPS administration (In the LPS model, both platelet-monocyte and platelet neutrophil aggregates increased at 3 hours, and decreased to levels lower than basal values at 6 hours from LPS injection (Analogously to what we observed in the LPS model, also in the CLP model platelet-monocyte adhesion was enhanced after sepsis induction in comparison to that measured in sham-operated mice , showingNo change in platelet-leukocyte adhesion was detected in mice injected with saline or mTPOR-MBP alone in both experimental models.Finally, the induction of systemic inflammation or sepsis was associated with a relevant decrease in leukocyte and platelet counts in both models, an effect that was neither observed in mice injected with saline or mTPOR-MBP alone, nor in sham-operated mice . The admThe aim of this study was to define the contribution of TPO in enhancing platelet-leukocyte adhesion associated with systemic inflammation and sepsis, and to explore the potential therapeutic effect of TPO blockade in inhibiting the development of organ damage in an acute endotoxemia model, which mimics a systemic inflammatory response syndrome, and in the CLP model, which is an abdominal infection model that leads to polymicrobial sepsis. Therefore, we synthesized a chimeric protein, mTPOR-MBP, and characterized it for its ability to inhibit in vivo the biological activity of TPO. We then tested its effects in these two widely used murine experimental models.Until now, the involvement of TPO in the pathogenesis of sepsis and other acute critical diseases has remained controversial. Whereas several observations have suggested, in recent years, that TPO may be involved in the pathogenesis of sepsis and of other critical diseases, for instance acute lung injury and acute pancreatitis , 15–18, In both experimental models, TPO and cytokine concentrations significantly increased after the induction of systemic inflammation or sepsis, confirming their resemblance, for this aspect, to what observed in patients , 16. FurThe administration of mTPOR-MBP inhibited the increase of platelet-monocyte binding both in the LPS model at three hours and in the CLP model, definitively showing that high TPO levels represent a pivotal stimulus of platelet activation and platelet-monocyte aggregation in these two models. On the contrary, mTPOR-MBP administration was associated with the persistence of platelet-neutrophil aggregates in the circulation at levels higher than in untreated mice, both in the LPS model at 6 hours and in the CLP model. Several studies have previously shown that platelet activation and thrombocytopenia are associated with the development of organ dysfunction during sepsis and with increased sepsis severity –37. FreqIn our study, we observed a dramatic reduction in leukocyte and platelet counts in both models, which was not prevented by mTPOR-MBP. Therefore, this well-described phenomenon is not related to TPO activity, but rather to the direct action of LPS on leukocytes, and, may be, platelets themselves. However, this observation excludes that the decrease in platelet-leukocyte aggregation we detected with mTPOR-MBP may be caused by the drop in leukocyte and platelet absolute counts.Quite surprisingly, mTPOR-MBP was not able to inhibit the increase in plasma concentrations of TNF-α, IL-6 and IL-10 observed in both models, a well-known phenomenon that participates in the initiation and progression of organ damage during systemic inflammation and sepsis . TherefoWe cannot precisely define the origin of the rise in TPO levels observed in our study after the induction of acute endotoxemia and polymicrobial sepsis. Platelet mass is considered the primary regulator of TPO levels , and botSeveral limits have to be taken into account in interpreting our results. First, we did nor evaluate the effect of TPO inhibition on long-term organ protection, neither on survival and recovery in both models. Therefore, we cannot exclude that the observed effects of mTPOR-MBP administration are transient, and/or that TPO inhibition has not a significant impact on disease evolution. Second, we cannot provide a clear demonstration of the mechanistic effect of TPO in the pathophysiology of systemic inflammation or sepsis. Although intriguing, the link between the observed effects of TPO inhibition on platelet-leukocyte interaction and the development of inflammation- or sepsis-associated tissue damage in target organs still remains not definitely proven by our experiments.In conclusion, blockade of TPO biological activity significantly reduced the severity of organ damage in both experimental endotoxemia and polymicrobial sepsis. This protective effect is associated with the inhibition of the increase in platelet-leukocyte adhesion, but not of cytokine release, observed during systemic inflammation and sepsis. Our results support the need to further explore the possibility of inhibiting TPO in the intent to prevent or reduce organ damage in patients affected by systemic inflammatory response or sepsis.


P = 0.001) in European subgroup. No publication bias was detected. Our results showed that the SNP49 polymorphism of CTLA4 gene was related to increased risk of GO.Many studies have established that T-lymphocyte antigen-4 (CTLA4) is a susceptible gene for Graves' disease (GD). Also many studies showed the association between the CTLA4 exon-1 49A/G polymorphism and the risk of developing Graves' ophthalmopathy (GO) in GD patients. But those results were inconsistent. In recent years many new studies were published which helped to shed light on the relationship of CTLA4 SNP49 with GO. So we performed the meta-analysis to explore the association between the SNP49 and GO susceptibility in GD patients. Studies up to February 29, 2012, were searched by using PubMed. The odds ratio was used to evaluate the strength of the association. Altogether 12 case-control studies involving 2,505 participants were included in the meta-analysis. Results showed that the G allele was related to the increased risk of GO compared with the A allele under allelic genetic model (OR = 1.14, 95% CI: 1.14–1.72,  Graves' disease is a thyroid autoimmune disorder with 25–50% individuals having ophthalmopathy which is called thyroid-associated ophthalmopathy (TAO) or GO. The clinical manifestations of GO can mostly be explained by the increased volume of extraocular muscles and orbital connective tissues . The symHuman CTLA-4 gene, located on 2q33, encodes a molecule which plays an important role in the downregulation of CD28 interaction with the ligands on the surface of antigen-presenting cells (APCs). The important inhibitory role of CTLA-4 in T-cell function has made it be one candidate gene when exploring autoimmune diseases.Many reports have established the association of CTLA-4 with GD –9. The COne meta-analysis exploring the association of CTLA-4 gene polymorphism with GO was performed in 2006 which found no definite result . Many neWe searched PubMed up to February 29, 2012, for all possible publications on the association between the CTLA-4 49A/G polymorphism and GO. Search terms were used as follows: “Cytotoxic T-lymphocyte associated antigen-4 or CTLA4,” “Graves' disease,” and “ophthalmopathy.” We also manually searched all reference lists of the relevant articles for additional papers. Language was limited to English. All eligible studies met the following criteria: (1) case-control design investigating the associations of CTLA4 49A/G polymorphism and GO; (2) providing genotype distribution information in both cases and controls or provided odds ratio (OR) with 95% confidence interval (95% CI) . When a study reported results on different subpopulations, we treated each subpopulation as a separate comparison in the meta-analysis. For overlapping data, the most complete or recent study was included.Two authors (Pengfei Du and Changjiang Wang) independently extracted data and reached consensus on all items. The following information was sought from each report: authors, journal and year of publication, country of origin, selection and characteristics of GO cases and controls, demographics, ethnic group of the study population, eligible and genotyped cases and controls, and number of cases and controls for the genotype. For studies including subjects of different ethnic groups, data were extracted separately for each ethnicity whenever possible.Z test was used to determine the significance of the pooled OR and P value <0.05 was considered significant. The between-study heterogeneity across all eligible comparisons was estimated using the chi-square based Q statistic [P < 0.10. Data were combined using both fixed-effects (Mantel-Haenszel) and random effects (DerSimonian and Laird) models [τ correlation coefficient) [P values were two-sided.The primary analysis compared GO cases with controls for the frequency of G versus A alleles. This analysis aims to detect overall differences. Pooled OR with 95% CI was used to evaluate the strength of the associations of CTLA-4 49A/G polymorphism and GO. When zero events occurred, we treated this problem by adding 0.5 to all the 2 × 2 cells of the contingency table to calculate the study-specific OR just as implemented in RevMan software. We examined the association of 49A/G polymorphism and GO for allelic contrast (G versus A). Furthermore, we conducted subgroup analyses by stratifying ethnicity into Europeans and Asians separately. A tatistic . Heterog) models . Random ) models . Inverteficient)  evaluateThe primary literature search yielded 17 papers , 28, 29.P heterogeneity = 0.01), and the random effects pooled OR was not significant: OR = 1.14, 95% CI: 0.94–1.38, P = 0.18  [P = 0.002, P heterogeneity = 0.32)  . The pooled frequency of G allele was 68.1% and 51.8% for cases and controls, respectively. Two studies suggested an at-risk effect of G allele , 24 and P = 0.18 . One stu = 0.19) . Sensiti = 0.32) . The G c = 0.01) .The current meta-analysis including 12 case-control studies was in an effort to clarify the relationship between CTLA-4 gene polymorphism and GO susceptibility. The overall results indicated that the CTLA-4 49A/G polymorphism was associated with susceptibility of GO which was more significant in European population. Although obvious heterogeneity was detected for 49A/G associations, sensitivity analyses did not materially alter the overall and subgroup results under different genetic models, indicating that the results were stable and reliable.The polymorphism of CTLA-4 had been suggested in many diseases which included Addison's disease, autoimmune hypothyroidism, and rheumatoid arthritis. The CTLA-4 49A/G SNP in exon-1 leads to the substitution of Ala with Thr in the signal peptide part which was reported to cause misprocessing of CTLA-4 in the ER resulting in less efficient glycosylation and diminished surface expression of CTLA-4 protein . StudiesThe association of CTLA-4 49A/G SNP with GO was more significant in European population but not in Asian population. The CTLA4 49A/G SNP shows the same effect on the GO development with different significance. The results are confusing but not conflicting. This can be explained by the following reasons. Firstly, both GD and GO are multigenic conditions. It is unlikely to have one major susceptibility gene contributing to the development of GO. In the studies of Xu et al., they transiently transfected a T-cell line with a CTLA-4 construct harboring either the G or the A allele of the 49A/G SNP and found no difference in CTLA-4 expression and/or function harboring the A or the G allele . It is lIn conclusion, our analysis first intensified the association between 49A/G and GO susceptibility, which showed that the G allele is a risk factor for GO. More risk factors should be considered in future association studies of the CTLA-4 SNP with GO. The gene-gene and gene-environment interaction should also be taken into consideration in future research. The identification of the candidate gene and understanding of the mechanisms by which they cause disease will identify those individuals at risk for developing GO in the future. Treatment will evolve towards more preventive strategies. The treatment will be personalized by targeting the specific pathways that are most contributory to GO development. We believe that the future will witness the engagement of novel molecules, such as altered peptide ligands  and spec


Balance and maintaining postural equilibrium are important during stationary and dynamic movements to prevent falls, particularly in older adults. While our sense of balance is influenced by vestibular, proprioceptive, and visual information, this study focuses primarily on the vestibular component and its age-related effects on balance. C57Bl/6J mice of ages 1, 5–6, 8–9 and 27–28 months were tested using a combination of standard (such as grip strength and rotarod) and newly-developed behavioral tests . In the current study, we confirm a decline in fore-limb grip strength and gross motor coordination as age increases. We also show that a vestibular stimulus of low frequency (2–3 Hz) and duration can lead to age-dependent changes in balance beam performance, which was evident by increases in latency to begin walking on the beam as well as the number of times hind-feet slip (FS) from the beam. Furthermore, aged mice (27–28 months) that received continuous access to a running wheel for 4 weeks did not improve when retested. Mice of ages 1, 10, 13 and 27–28 months were also tested for changes in walking trajectory as a result of the vestibular stimulus. While no linear relationship was observed between the changes in trajectory and age, 1-month-old mice were considerably less affected than mice of ages 10, 13 and 27–28 months. Conclusion: this study confirms there are age-related declines in grip strength and gross motor coordination. We also demonstrate age-dependent changes to finer motor abilities as a result of a low frequency and duration vestibular stimulus. These changes showed that while the ability to perform the balance beam task remained intact across all ages tested, behavioral changes in task performance were observed. It is well documented that advancing age is a significant risk factor for falls. Loss of balance and subsequent falls often lead to deleterious effects including repeated falls, anxiety, admission into high care facilities such as nursing homes and hospitals, and in some cases death in older adults , vestibulo-collic (VCR) and vestibulo-spinal reflexes (VSR). The reflexes can be used in the diagnosis of vestibular impairment  is not sufficient to mitigate these age-related changes.ad libitum. Prior to tests, mice were brought into the testing room for a 10 min acclimatization period.All procedures outlined were approved by the Animal Ethics Committee at The University of Sydney. C57BL/6J mice  between 1 and 29 months were tested on the behavioral tasks described below. All mice were held at The University of Sydney Bosch Rodent Facility on a 12 h light/dark cycle with access to food and water Mice were tested using a battery of balance and motor performance tasks as described below.The forelimb grip strength of mice was measured using a Digital Force Gauge . Mice were held by the base of the tail close to the horizontal bar (attached to the force gauge) to allow them to reach and grab onto the bar with their forelimbs. Mice were then positioned so that their body was horizontal and in line with the bar. They were then pulled horizontally away from the bar by the tail until their grip was released. The tension was measured and defined as grip strength. Mice were given 1 min inter-trial intervals (ITI) during which they were returned to their cages with access to food and water. This procedure was repeated for a total of five trials for each mouse . All grip strength values acquired were normalized for body weight.Motor performance was assessed using the protocol described in Tung et al. . BrieflyMore subtle motor coordination and balance was assessed using a modified balance beam test  to the opposite corner where a darkened goal box was located. The floor of the arena was lined with tracing paper. Training was carried out in a similar manner to that used for balance beam experiments, where mice were progressively placed further away from the goal box and towards the starting location until they were able to walk 30 cm unassisted from the starting position to the goal box. Using a cotton tip, petroleum jelly (Vaseline) was applied to the soles of the mouse’s hind-feet. Mice were then placed at the starting position so that they could walk to the goal box. This was repeated in instances where mice did not walk directly to the goal box or required encouragement by prodding, and was repeated until mice performed the task unassisted. The unassisted trial was used for analysis. Mice then underwent the vestibular stimulus (as above) and were immediately placed back at the starting position to perform the task. During the testing of 1-month-old mice, an additional light source was used to encourage mice to walk towards the goal box.For analysis, graphite powder (Pressol) was used to color the petroleum jelly residue on the tracing paper  was added to each cage of 27–28-month-old mice. The running wheel remained in the cages for 4 weeks and were wirelessly connected to a PC in order to monitor running wheel usage in each cage of mice. After this period, mice were retested on all behavioral tasks.t-test or ANOVA, or their non-parametric equivalent, and significance was set at p < 0.05. Either Tukey or Bonferroni post hoc tests were used for multiple comparisons. Values are reported as mean ± SD.IBM SPSS Statistics software (Version 21) was used for statistical analysis. Unless otherwise stated, analysis was conducted using either two-tailed Student’s F = 6.727, p = 0.001). A post hoc test showed that both 1 (4.44 ± 0.74 g/g) and 5–6-month-old mice (4.36 ± 0.75 g/g) were stronger than 8–9-month-old mice  and 27–28-month-old mice  11.431, p = 0.01). When individual age groups were compared, a number of differences were observed. First, post hoc analysis showed that 8–9-month-old mice (13.81 ± 2.86 s) displayed shorter TTF when compared with 1-month-old mice  and 5–6-month-old mice  5.948, p = 0.114). In contrast, baseline measurements of TTT  19.652, p < 0.001) and FS  15.120, p = 0.002) both showed increases with age.Three different measures were used to assess changes in balance beam performance across all age groups: TTS, TTT and number of foot slips (FS). These measurements were recorded both before and after the vestibular stimulus Table . BaselinF = 3.573, p = 0.027), with the post hoc test showing 1 (p < 0.01) and 5–6-month-old (p < 0.05) mice were affected by the stimulus differently to 27–28-month-old mice  = 4.572, p = 0.010; Figure Post hoc tests revealed greater increases in TTT and FS for 27–28-month-old mice when compared to 1 (ps < 0.001), 5–6 (ps < 0.001), and 8–9-month-old mice (ps < 0.001).Following the vestibular stimulus an age-related increase in measurements of TTS was observed  were tested on a smaller (9.5 mm diameter) beam using the same balance beam protocol and compared with 1-month-old control mice reported above (n = 9). No differences in baseline measurements were observed due to the different beam diameters. Furthermore, the different beam diameters did not result in changes in balance beam measures following the vestibular stimulus.Due to their age, 1-month-old mice have comparably smaller gait widths than the older mice. Therefore, to assess whether a smaller gait width to beam diameter ratio resulted in better balance beam performance in younger mice, 1-month-old mice (n = 6) and without (n = 6) the darkened vestibular chamber, no differences were shown for all balance beam measures. Additionally, when 1 (n = 6) and 8–9-month-old mice (n = 6) were tested using the darkened chamber, no age-related changes were evident, suggesting the results described above are independent of visually-mediated effects.To determine whether visual input during the vestibular stimulus influenced balance beam outcomes , black contact paper was used to line the interior surface of the vestibular stimulus chamber. When comparing 8–9-month-old mice with (p = 0.001), with 1-month-old mice having much smaller minor axis length than 10 (p = 0.001), 13 (p < 0.01), and 27–28-month-old mice . Instead, post hoc tests revealed that changes in minor axis length in 1-month-old mice as a result of undergoing the vestibular stimulus (before: 6.90 ± 1.66 mm vs. after: 6.64 ± 1.85 mm), were significantly different from those observed in 10 , 13 , and 27–28-month-old mice  and no changes in grip strength or TTF were observed as a result of adding a running wheel into home cages.; Mann-Whitney U = 7, p = 0.029). The running wheel did not improve balance beam measures post-vestibular stimulus. There was also no improvement in walking trajectory observed as a result of adding a running wheel.During balance beam tests, three of the eight 27–28-month-old mice fell during the trial prior to the vestibular stimulus and four mice fell after the stimulus (as above). Two of these mice fell before and after the stimulus and were excluded from further testing due to health problems. At 29 months, after a running wheel was added to home cages for 4 weeks, three of the six mice fell in the trial prior to the stimulus. Two of these mice also fell after the vestibular stimulus, and had fallen during testing at 27–28 months of age. There were no changes in baseline measurements of TTS and TTT as result of the running wheel, though there was an increase in the number of FS observed at 29 months of age . Other studies have used centrifuges to induce motion sickness in rats, and have also measured consumption of either saccharin . No differences were observed in the performance of young mice between the small and large beams. Further, stimulation of the vestibular system with the rotatory stimulus did not alter performance on the smaller beam. This suggests that deterioration in balance (with and without stimulation of hair cells) as age increases is not due to changes in gait width or weight which has the potential to hinder the ability for mice to grip onto the beam  mice for 4 weeks. We observed an increase in the number of FS prior to the vestibular stimulus, and no improvements in performance after the vestibular stimulus that were attributable to the running wheels. A previous study has reported running wheels improve the motor coordination of younger mice. Mice housed with running wheels after weaning and tested at approximately 3 months of age scored better in the accelerating rotarod test than mice given a locked running wheel , and the central vestibular components that form the output of the balance system.VWKT, TJB, SLQ, MAM, and AJC were involved in developing and conducting experiments, data analysis, and writing and/or editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


INK4A. To identify age-related molecular factors, we performed microarray and gene ontology analyses. These analyses revealed an intriguing transcriptomal shift in A-TSPC, where the most differentially expressed probesets encode for genes regulating cell adhesion, migration, and actin cytoskeleton. Time-lapse analysis showed that A-TSPC exhibit decelerated motion and delayed wound closure concomitant to a higher actin stress fiber content and a slower turnover of actin filaments. Lastly, based on the expression analyses of microarray candidates, we suggest that dysregulated cell–matrix interactions and the ROCK kinase pathway might be key players in TSPC aging. Taken together, we propose that during tendon aging and degeneration, the TSPC pool is becoming exhausted in terms of size and functional fitness. Thus, our study provides the first fundamental basis for further exploration into the molecular mechanisms behind tendon aging and degeneration as well as for the selection of novel tendon-specific therapeutical targets.Although the link between altered stem cell properties and tissue aging has been recognized, the molecular and cellular processes of tendon aging have not been elucidated. As tendons contain stem/progenitor cells (TSPC), we investigated whether the molecular and cellular attributes of TSPC alter during tendon aging and degeneration. Comparing TSPC derived from young/healthy (Y-TSPC) and aged/degenerated human Achilles tendon biopsies (A-TSPC), we observed that A-TSPC exhibit a profound self-renewal and clonogenic deficits, while their multipotency was still retained. Senescence analysis showed a premature entry into senescence of the A-TSPC, a finding accompanied by an upregulation of p16 MSC are capable of mobilizing, proliferating, and committing to terminally differentiated cell types. Thus, MSC are a potent cell source for tissue regeneration  within human hamstring tendons. They showed that the TSPC exhibit classical MSC characteristics, having typical surface antigens, self-renewal, clonogenicity, and three-lineage differentiation capacity. Yet, in contrast to MSC, TSPC express tendon-related genes such as scleraxis and tenomodulin and are able to form tendon and enthesis-like tissues when implanted in vivo. The existence of TSPC was further confirmed in subsequent studies with tendons from different species  with those of aged patients with functionally degenerated tissues (A-TSPC). Using microarray analysis and different functional readouts, we investigated whether TSPC alter their molecular and cellular characteristics during aging and degeneration. For the first time to our knowledge, this study identifies the distinct molecular and cellular changes in TSPC that contribute to human tendon aging and degeneration.in vitro to validate their stem/progenitor character. We used FACS and immunocytochemistry to examine the expression of surface antigens and stem cell markers in TSPC based on the studies by Bi et al.  and characterized i et al. , Tempfer  staining Fig.E,F. AlreINKA4 upregulation.Taken together, our results clearly indicate that A-TSPC have a reduced self-renewal potential, which was apparent during short- and long-term cultivation but also when the cells were stimulated with FGF-2 or TGF-β1. We suggest that this phenomenon is due to an earlier entry of A-TSPC into cellular senescence, which we confirmed by demonstrating that the A-TSPC group exhibited a significantly higher number of senescent cells along with a substantial p16Multilineage differentiation is another feature of stem/progenitor cells. Therefore, we compared TSPC capacity to undergo adipogenic, osteogenic, and chondrogenic differentiation . Regarding adipogenic and osteogenic differentiation, there were no obvious differences between the groups and the individual donors. Regarding chondrogenic differentiation, pronounced donor variability was observed in each group; however, both Y- and A-TSPC produced proteoglycan-rich matrices and the cartilage marker aggrecan. In summary, we concluded that TSPC multipotency is not strongly impaired upon aging.To identify molecular factors involved in tendon aging and degeneration, we performed microchip hybridization with RNA from three different donors per group. Comparative microarray analysis is summarized in a Venn diagram in Fig.For the first time to our knowledge, our microarray analysis of patient-derived TSPC reports transcriptomal alterations that are specific for tendon aging and degeneration. Moreover, it strongly suggests that A-TSPC will exhibit further phenotypic and behavioral differences to Y-TSPC. Therefore, in the following part of the study, we compared the migratory activity, actin organization, and turnover as well as the expression of several microarray gene candidates in Y- and A-TSPC.in vitro scratch assay mimicking wound closure. Quantifications of migratory distance revealed that A-TSPC migration speed and distances were significantly slower compared with Y-TSPC  collagen I and fibronectin, which are essential for cell adhesion and migration; (ii) the collagen I-binding integrins α1β1, α2β1, and α11β1 and the fibronectin-binding α5β1, αvβ3, and αvβ5  not only offered new insights into the biology of tendon cells but also demonstrated that distorted TSPC functions can be linked to the progression of tendon pathologies such as tendinous ossification  to determine the TSPC self-renewal and differentiation capacity by serial transplantation assays in vivo.Regarding the influence of aging on stem cells multipotency, there are very controversial results. Some studies showed that MSC from aged tissue samples exhibit a tendency to reduce the degree of three-lineage differentiation  of the A-TSPC cohort, which revealed very comparable morphological and cell growth characteristics, 3–4 TSPC strains were selected as representative and employed in all experiments. Unless otherwise stated in the figure legends, TSPC in passages 1–5 (PD 2–10) were used in the experiments.We obtained cells from human nonruptured Achilles tendons from 16 patients, who had undergone diverse surgical operations in the Surgical Clinic of Ludwig Maximilians University in Munich (LMU). The procedure was approved by the Ethical Commission of the LMU Medical Faculty (grant No. 166-08), and informed consent was obtained from all donors. The samples were separated into two groups based on the clinical signs and pathological evaluation: young and healthy (abbreviated as Y-TSPC) and aged and degenerated (abbreviated as A-TSPC) (Table S1). The Y-TSPC group consisted of four donors with an average age of 28±5 years, while the A-TSPC group consisted of 12 donors with an average age of 63±14 years. One 37-year-old donor was included in this group because of clear clinical signs of tendon degeneration. TSPC were isolated according to Bi et al. : the tenThe protocols and antibodies used in the study are provided in the supporting information.et al. (3 cells per cm2) were grown in 48- or 96-well plates for 24 h. Next, cells were incubated in complete culture medium containing 0.2% FBS, with or without FGF-2 , or TGF-β1  for 7 or 3 days, respectively. Thereafter, WST-1 reagent was applied according to the manufacturer's instructions . Optical density (OD) was measured at 420 nm and 620 nm using a Microtiter Reader . The average OD value of Y-TSPC donors was set as control–100%.Cumulative population doubling (PD) and colony-forming unit (CFU) assay were carried out as described in the study by Alberton et al.  and the 3 cells per cm2) from passages 4 , 12 , and 14  were grown in 12-well plates for 72 h, then fixed, and incubated with the kit's staining mixture for 16 h at 37 °C. Photographs were taken with AxioCamICc3 color camera on AxiovertS100 . In four random dish areas, β-gal-positive cells were expressed in percentage versus the total number of cells.β-Galactosidase  activity assay was performed using the Senescent Cells Staining Kit (Sigma-Aldrich). TSPC  and 5  toward adipogenic, osteogenic, and chondrogenic lineages according to Alberton et al.  and the et al. , and individual cell tracks were analyzed with ImageJ V1.38 software. For scratch assay, 1 × 104 cells per cm2 were plated on collagen I  or fibronectin -coated 6-well dishes and were let to form confluent cell layers for 48 h. Prior imaging, the layers were scratched multiple times. Time-lapse was performed with 4 frames per h for 72 h. For each donor group, the initial length of four scratches and the time for complete scratch closure were measured and used for calculation of cell velocity.An automated inverted microscope AxiovertS100 equipped with AxioCamMRm camera (Carl Zeiss) and biochamber  was used. For analysis of random migration, 1.5 × 103 cells per cm2) were plated for 48 h on 96-well plates. Latrunculin A  was diluted to 0.4 μm in complete culture medium containing 0.2% FBS and applied in a time-dependent manner. Afterward, the cells were fixed in 3.7% formalin/PHEM  solution and permeabilized with 0.1% Triton X-100, and their F-actin was stained with phalloidin-AF546  as described in Docheva et al. . For F-actin analysis, TSPC  were seeded in 6-well dishes. After 3 days, the conditional medium, supplemented with protease inhibitors (Roche), was collected, and the cell number was counted. Secreted collagen type I was determined using Human Collagen I ELISA kit  according to the manufacturer's instructions. OD was measured at 450 nm on a Microtiter Reader (Thermo Scientific). The collagen I concentration was calculated using collagen I standard curve and normalized to the cell number. The production of fibronectin was measured with Human Fibronectin ELISA kit (Millipore) following the manufacturer's recommendations and using 10 μg RIPA protein extracts from TSPC. The OD was measured at 450 nm, and the fibronectin concentration was interpolated from the kit's standard curve. ROCK2 activity was assessed by ROCK Activity kit . The protein input and OD readout are the same as in the fibronectin ELISA kit.Western blotting protocols are given in the supporting information. For ELISA assays, TSPC  for 72 h. Media was changed every day to ensure active ROCK inhibition. Treated A-TSPC were subjected to quantification of cell area, analyses of actin organization, content and turnover, and evaluation of migration ability as described above. Nontreated Y-TSPC and A-TSPC were used in parallel for comparison.n = 6) or three independent experiments (n = 9), each performed in duplicates or triplicates. Bar charts represent means and standard deviations. For testing statistical significance, unpaired Student's t-test was used. A P-value <0.05 was considered statistically significant and it was indicated in the figures as follows *P < 0.05, **P < 0.005, ***P < 0.0005.Quantitative data and statistical significance were analyzed using GraphPad Prism 5.0 software . Experiments were performed with minimum six different donors (three different donors per group). All quantitative data were acquired from two (